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R R Kay

Publications and source records attributed to R R Kay.

At least 55 records · Page 3Linked to original sources
PubMed1988

Structure elucidation of two differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime mould Dictyostelium discoideum.

Two endogenous differentiation-inducing factors (DIF-2 and DIF-3), which induce stalk-cell differentiation in the cellular slime mould Dictyostelium discoideum, have been identified as the pentan-1-one and monochloro analogues respectively of (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one). These compounds represent a new chemical class of effector molecules.

Cell Differentiation↗
PubMed1988

Signals controlling cell differentiation and pattern formation in Dictyostelium.

The major inducers of cell differentiation in Dictyostelium appear to be cyclic AMP and DIF-1. Recently we have chemically identified DIF-1, together with the closely related DIF-2 and -3. They represent a new chemical class of potent effector molecules, based on a phenyl alkanone with chloro, hydroxy, and methoxy substitution of the benzene ring. Previous work has shown that DIF-1 can induce prestalk-specific gene expression within 15 min, whereas it suppresses prespore differentiation. Hence, DIF-1 can control the choice of pathway of cell differentiation in Dictyostelium and is therefore likely to be involved in establishing the prestalk/prespore pattern in the aggregate. In support of this, we show that DIF treatment of slugs results in an enlarged prestalk zone. Cyclic AMP seems less likely to have such a pathway-specific role, but later in development it becomes inhibitory to stalk cell differentiation. This inhibition may be important in suppressing terminal stalk cell differentiation until culmination. Spore differentiation can be induced efficiently by high levels of Br-cyclic AMP, a permeant analogue of cyclic AMP. In this, it phenocopies certain spore-maturation mutants, and we propose that during normal development spore differentiation is triggered by an elevation in intracellular cyclic AMP levels. How this elevation in cyclic AMP levels is brought about is not known. The experiments with Br-cyclic AMP also provide the first direct evidence that elevated levels of intracellular cyclic AMP induce differentiation in Dictyostelium.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum.

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.

Cell Differentiation↗
PubMed1987

Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen.

We have isolated a gene that is very rapidly induced at the transcriptional level by DIF--a low molecular weight, diffusible factor necessary for stalk cell differentiation in Dictyostelium cells developing in vitro. The gene encodes a protein containing an N-terminal signal peptide preceding approximately 70 tandem repeats of a highly conserved 24 amino acid sequence with a high cysteine content. These features suggest it is an extracellular structural protein. During normal development, the gene is maximally expressed in the slug, in which the mRNA is very highly enriched in prestalk over prespore cells. The gene is not detectably expressed until the tipped aggregate stage, several hours later than prespore genes, suggesting that prespore cell differentiation precedes prestalk cell differentiation. The demonstration that DIF induces a gene normally only expressed in the prestalk zone of the slug provides strong evidence that DIF is a Dictyostelium morphogen.

Amino Acid Sequence↗
PubMed1987

Nature and distribution of the morphogen DIF in the Dictyostelium slug.

The Dictyostelium slug contains a simple anterior-posterior pattern of prestalk and prespore cells. It is likely that DIF, the morphogen which induces stalk cells, is involved in establishing this pattern. Previous work has shown that a number of distinct species of DIF are released by developing cells and that cell-associated DIF activity increases rapidly during the slug stage of development. In this paper we describe a comparison of the DIF extracted from slugs with the DIF released into the medium. Analysis by high-pressure liquid chromatography (HPLC) using different solvent systems shows that the major species of DIF activity extracted from slugs coelutes with DIF-1, the major species of released DIF and is similarly sensitive to sodium borohydride reduction. Since DIF specifically induces the differentiation of prestalk cells, the anterior cells of the slug, it could be anticipated that DIF is localized in the prestalk region. We have therefore determined the distribution of DIF within the slug. Migrating slugs from strain V12M2 were manually dissected into anterior one-third and posterior two-third fragments and the DIF activity extracted. Surprisingly, we found that DIF was not restricted to the prestalk fragment. Instead there appears to be a reverse gradient of DIF in the slug with at least twice the specific activity of total DIF in the prespore region than in the prestalk region.

Animals↗
PubMed1987

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum.

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.

Base Sequence↗
PubMed1986

Intracellular pH in Dictyostelium: a 31P nuclear magnetic resonance study of its regulation and possible role in controlling cell differentiation.

Intracellular pH (pHi) has been measured in Dictyostelium discoideum cells by 31P nuclear magnetic resonance. Ax2 cells, newly harvested from growth medium, maintained a pHi of 7.33 +/- 0.04 (17) at an extracellular pH ranging from 3.5 to 6.5. Below pH 3.5 the cells tend to lyse, whereas at pH values above 6.5 their pHi rises though they remain viable. pHi regulation in acid medium is not dependent on external Na+ or any other inorganic ion and so most probably involves the electrogenic plasma membrane proton pump. No significant change in pHi was detected during development through to the slug stage. Mature stalk cells gave a very acidic phosphate signal (pH less than or equal to 5.5) which was probably vacuolar in origin. Indirect experiments had suggested that pHi might regulate the development of Dictyostelium cells, with low pHi favouring stalk cell and high pHi favouring spore cell differentiation. In particular, two inhibitors of the plasma membrane proton pump, diethylstilbestrol and zearalenone, had been shown to be stalk cell inducers. In the present studies measurements of pHi of cells exposed to these inducers failed to detect the expected drop in pHi. In addition, DIF-1 (a low Mr factor), the natural inducer of stalk cell formation, caused, if anything, a slight alkalinization of the cells. Thus the original theory linking pHi and cell differentiation is not supported by these results and therefore appears to require some modification. Finally, extract experiments revealed the existence of two unidentified abundant phospho-compounds with resonant frequencies close to inorganic phosphate. The existence of these compounds can complicate the interpretation of spectra gained from living Dictyostelium cells.

Cell Differentiation↗
PubMed1985

Selective induction of stalk-cell-specific proteins in Dictyostelium.

We compared the proteins synthesized and accumulated by Dictyostelium discoideum amoebae in response to the morphogenetic factor termed differentiation-inducing factor (DIF) to assess the proposed ability of DIF to regulate the choice of differentiation pathway. When amoebae of a mutant strain with low endogenous DIF levels were given DIF, they dramatically increased the expression of 21 of 23 proteins preferentially found in stalk cells, but drastically repressed 4 major spore-specific proteins. Most of the induced proteins were also expressed in amoebae of a developmentally competent strain developing at low cell densities and exposed to DIF, low extracellular pH, or the proton pump inhibitor diethylstilbestrol; this suggests that an intracellular acidification may be a key part of the mechanism of DIF action. We conclude from the similar morphology and extensive homology of proteins of DIF-induced and stalk cells that most stalk-pathway functions are DIF dependent.

Animals↗
PubMed1983

Purification of stalk-cell-inducing morphogens from Dictyostelium discoideum.

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low-Mr factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262, 717-719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76, 308-312]. These differentiation-inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD-2 resin, extraction into hexane and two steps of reverse-phase high-pressure liquid chromatography (HPLC). Our results show the following. HPLC resolves a major stalk-cell-inducing activity (DIF-1) and at least four minor and more polar activities (DIFs 2-5). DIF-1 has been purified at least 3000-fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF-1 can be explained in part by the loss of non-specific stimulatory ('helper') factors during the purification. A few micrograms purified DIF-1 were obtained from 10(12) cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM. The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF-5 was partially sensitive and DIF-4 resistant. Other properties of DIF-1 suggest that it is a non-polar molecule of Mr less than 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties. The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.

Biological Assay↗
PubMed1983

Dictyostelium mutants lacking DIF, a putative morphogen.

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.

Cell Differentiation↗
PubMed1982

cAMP and spore differentiation in Dictyostelium discoideum.

The individual amoebae composing of Dictyostelium aggregate can differentiate into either stalk or spore cells according, it is believed, to the extracellular signals they receive. By inducing the differentiation of isolated cells we hope to identify these signals. It is shown here that wild-type cells can differentiate into prespore cells in a solution of cAMP plus salts supplemented by conditioned medium. Cell-to-cell contact is not required. More important, isolated cells of various sporogenous mutant strains form spores in similar conditions without needing conditioned medium at all. For these strains at least, cAMP is the sole inducer necessary for spore formation. Earlier work has shown that stalk cells are induced by a combination of cAMP and a low molecular weight factor, differentiation inducing factor (DIF). DIF now appears to be the only pathway-specific inducer involved in the differentiation of sporogenous amoebae and DIF levels in the aggregate may therefore determine whether an amoeba becomes a stalk or a spore cell. In suitable conditions some sporogenous mutants form migrating slugs having an anterior/posterior pattern of prestalk and prespore cells. This pattern could be generated by the localized activity of DIF.

Cell Aggregation↗
PubMed1981

Cell patterning in Dictyostelium.

We summarize studies on stalk and spore cell formation in D. discoideum cell monolayers, aimed at revealing factors involved in controlling the prestalk:prespore pattern in this organism. We propose that there are no cell interactions dependent on cell contact per se. Formation of mature stalk cells from isolated amoebae incubated in a buffered salts medium requires only cyclic AMP and a lipid-like factor (DIF) released by cells developing at high density. In addition, a variety of sporogenous mutants can form spores rapidly and efficiently when incubated at low density in tissue culture dishes containing a similar cyclic AMP and salts medium. In some cases spore formation is improved by the addition of one or other of a variety of protective agents such as bovine serum albumin. Wild-type amoebae at low density form prespore cells under the same conditions. We present some evidence that DIF is the activator of prestalk cell formation in a two-component patterning mechanism of the kind proposed by Wolpert et al. (Symp. Soc. exp. Biol. 25, 391-415 (1971)) and Gierer & Meinhardt (Kybernetik 12, 30-39 (1972)). We also provide data indicating that the role of inhibitor is played by ammonia, an idea first mooted by Sussman & Schindler (Differentiation 10, 1-5 (1978)).

Cell Adhesion↗
PubMed1981

Dictyostelium amoebae can differentiate into spores without cell-to-cell contact.

Amoebae of sporogenous mutants of Dictyostelium discoideum can differentiate into stalk cells and spores in the absence of normal morphogenesis when spread on agar containing cyclic-AMP. The efficiency of differentiation is improved when the amoebae are incubated as submerged monolayers in plastic petri dishes. Under these conditions spore formation is density dependent and hence requires some form of cellular interaction. To determine whether this interaction involves direct cell-cell contact we have made time-lapse films of cells differentiating at intermediate density. These films show that amoebae can develop into spores without making contact with any other cells. In addition, although some cells do divide during incubation, division is not necessary for spore formation. At higher densities small aggregates form which give rise to mixtures of stalk cells and spores. There is no detectable patterning of the two cell types within such aggregates.

Dictyostelium↗
PubMed1979

Gene expression in Dictyostelium discoidium: mutually antagonistic roles of cyclic-AMP and ammonia.

Cyclic-AMP and ammonia have been previously identified as extracellular signals during Dictyostelium development. Both are important in controlling morphological movements and cyclic-AMP also in inducing gene expression. The work in tis paper is concerned with their effects on developmental gene expression. Cyclic-AMP was found to act as an inducer during the aggregative (as exemplified by phosphodiesterase) and the post-aggregative (glycogen phosphorylase, UDP-galactose polysaccharide transferase, prespore vacuoles and stalk cells) phases of gene expression. Ammonia inhibited the appearance of each of the above markers and antagonized the inductive effects of cyclic-AMP on them. This inhibition by ammonia of cyclic-AMP inducible gene expression may involve a step linking elevated intracellular cyclic-AMP levels to gene activation. It has been suggested that the specification of cells within the aggregate into the stalk and spore pathways of differentiation might be controlled by cyclic-AMP and ammonia. In this model for pattern formation cyclic-AMP would induce stalk cell differentiation and ammonia spore formation. The present results argue against this idea since cyclic-AMP induces and ammonia inhibits differentiation along both pathways. The function of these agents may rather be to coordinate the rates of biochemical differentiation of individual cells and link them to the overall morphological changes occurring during development.

3',5'-Cyclic-AMP Phosphodiesterases↗